Mouse anti cut

For immunohistochemistry, embryos were fixed and stained according to standard procedures. Guinea pig anti-SIMU antibodies [42 (link)] was used at a 1:5000 dilution, while rabbit anti-Htl antibodies, a gift from T. Kojima, were used at a 1:1000 dilution. Rabbit anti-activated Dcp-1 (Cell signaling) and mouse anti-GFP (Roche) antibodies were used at a 1:100 dilution each. Mouse anti-REPO, mouse anti-Eve and mouse anti-CUT (Developmental Studies Hybridoma Bank) antibodies were used at 1:20, 1:100 and 1:5 dilutions, respectively. Fluorescent secondary antibodies (Cy3- and Cy5-labeled antibodies from Jackson ImmunoResearch and Alexa Fluor 488-labeled antibodies from Molecular Probes) were used at 1:200 dilutions each. A 75% glycerol solution was used as imaging medium. Microscope imaging and data analyses of all images were acquired on a Zeiss AxioImager M2 microscope equipped with an ApoTome2 optical sectioning device. All images taken using Zen acquisition software and processed with Adobe Photoshop CS6.

For fluorescent imaging, salivary glands, wing discs from different juvenile stages and pupal wings were dissected in 1× phosphate-buffered saline (PBS) and fixed in 4% formaldehyde for 20 min at RT. The tissues were rinsed in 0.1% Triton X-100 (PBST) or 0.3% PBST in pupal wings for 1 hr and incubated at 4°C with primary antibodies diluted in PBST overnight. After incubation with primary antibodies, the tissues were washed with PBST (3×10 min washes) and incubated with adequate combinations of secondary antibodies (Alexa Conjugated dyes 488, 555, 647, Life Technologies, 1:500) for 2 hr at RT, followed by 3×10 min washes with PBST, and then rinsed with PBS before mounting in Vectashield with DAPI (Vector Laboratories, H1200) for image acquisition. The following primary antibodies were used at indicated dilution: rat anti-Chinmo (1:500, N, Sokol), mouse anti-Cut (1:200, Developmental Studies Hybridoma Bank (DSHB) #2B10), mouse anti-Wg (1:200, DSHB #4D4), mouse anti Br-C core (1:250 DSHB #25E9.D7), rabbit anti-cleaved Dcp-1 (1:100, Cell Signaling #9578), and rabbit anti-E93 (1:50, this work).

Embryo fixation was performed according to standard methods. Embryos were fixed by a solution containing heptane (Sigma, St Louis, MO) and methanol^{57 (link),58 (link)}. Wing discs were fixed in PLP fixative (2% paraformaldehyde, 75 mM lysine, and 35 mM phosphate buffer, pH7.4) for 15–30 min at room temperature.
Antibodies used for immunohistochemistry were as follows: mouse anti-Cut (1:200, 2B10, Developmental Studies Hybridoma Bank (DSHB), Iowa City, Iowa), guinea pig anti-Centrosomin (1:1000, from Jordan Raff), Rat anti-α-Tubulin (1:200, MAB1864, Millipore, Burlington, Massachusetts), rabbit anti-PH3 (1:200, 06–570, Millipore), Rabbit anti-Vtd (1:500, this study), rabbit anti-Myc (1:100, ab9106, Abcam, Cambridge, UK). Secondary antibodies conjugated with Rhodamine Red™-X (RRX), Alexa Fluor® 647 or fluorescein isothiocyanate (1:200, 715-095-151, 715-295-151, 715-605-151, 711-095-152, 711-295-152, 711-605-152, 706-095-148, 706-295-148, 706-605-148, 712-095-153, 712-295-153, 712-605-153) were from Jackson ImmunoResearch Inc. Vectashield with 4′, 6-diamidino-2-phenylindole (H-1200, Vector Laboratories, Burlingame, CA) was used for mounting. Fluorescent images were acquired using Carl Zeiss LSM710 confocal microscope (Carl Zeiss, Oberkochen, Germany).